Field of the Invention
The present invention relates to systems and methods of temperature referencing for melt curve data collection. More specifically, the present invention relates to systems and methods for collecting DNA melt curve data for a DNA sample and a temperature reference material.
Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. PCR is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones. See, for example, Lagally et al. (Anal Chem 73:565-570 (2001)), Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (Anal Chem 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683), Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S. Patent Application Publication No. 2005/0042639).
Microfluidic systems are systems that have at least one channel through which a fluid may flow, which channel has at least one internal cross-sectional dimension, (e.g., depth, width, length, diameter) that is less than about 1000 micrometers. Typically, microchannels have a cross-sectional dimension of about 5 microns to about 500 microns and a depth of about 1 micron to about 100 microns.
Melt curve analysis is an important technique for analyzing nucleic acids. In this method, a double stranded nucleic acid is denatured in the presence of a dye that indicates whether the two strands are bound or not. Examples of such indicator dyes include non-specific binding dyes such as SYBR® Green I, whose fluorescence efficiency depends strongly on whether it is bound to double stranded DNA. As the temperature of the mixture is raised, a reduction in fluorescence from the dye indicates that the nucleic acid molecule has melted, i.e., unzipped, partially or completely. Thus, by measuring the dye fluorescence as a function of temperature, information is gained regarding the length of the duplex, the GC content or even the exact sequence. See, for example, Ririe et al. (Anal Biochem 245:154-160, 1997), Wittwer et al. (Clin Chem 49:853-860, 2003), Liew et al. (Clin Chem 50:1156-1164 (2004), Herrmann et al. (Clin Chem 52:494-503, 2006), Knapp et al. (U.S. Patent Application Publication No. 2002/0197630), Wittwer et al. (U.S. Patent Application Publication No. 2005/0233335), Wittwer et al. (U.S. Patent Application Publication No. 2006/0019253) and Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421).
A number of commercial instruments exist that perform thermal melts on DNA. Examples of available instruments include the Idaho Technology HR-1 high resolution melter and the Idaho Technology LightScanner high resolution melter. The HR-1 high resolution melter has the highest resolution fluorescent signal to noise ratio and temperature resolution of any commercial device. However, it suffers from a limitation that it can only analyze one sample at a time, and the sample container must be replaced manually. Replacement of the container for each test perhaps contributes to run-to-run temperature variability. The LightScanner high resolution melter also has good signal and temperature resolution, and operates on a 96-well plate sample container. However, it suffers from significant (˜0.3° C.) temperature gradients across the entire plate, as do other systems based on standard well plates. A typical mode of operation for these analyzers is to apply heat to the sample(s) in a controlled manner to achieve a linear rise in temperature versus time. Simultaneously, a stable continuous fluorescence excitation light is applied, and emitted fluorescence is collected continuously over fixed integration time intervals. The fluorescence intensity data is converted from a time basis to a temperature basis based on the knowledge of the temperature ramp versus time.
One of the difficulties inherent to this method is that the temperature control system has limited precision and accuracy. For example, the feedback signal used to control the heater may come from a temperature sensor that is physically displaced from the sample. During a temperature ramp, heat diffuses from the heat source to the sample through the sample container, and hence temperature gradients exist within the sample and across the sample container as well. A temperature sensor outside the sample container, even if perfectly accurate, will report a temperature that is offset somewhat from the instantaneous sample temperature. Furthermore, this offset will depend upon the ramp rate, the geometry and the quality of thermal contact between the heater and the sample container.
There is current market interest in further developing microfluidic genomic sample analysis systems for detecting and analyzing DNA sequences. The development of these microfluidic systems often entail the various combinations of channel configurations, inlets, outlets, buffer insertion methods, boluses of genomic sample insertion methods, temperature cycling and control methods, and optical analysis methods. There is also further interest in developing systems and methods for temperature referencing for melt curve data collection.
Microfluidic melting curve analysis is typically carried out either in a stopped flow format or in a continuous flow format. In a stopped flow format, flow is stopped within a microchannel of a microfluidic device while the temperature in that channel is ramped through a range of temperatures required to generate the desired melt curve. A drawback to the stopped flow format is that it does not integrate well in systems with other flow through processes which require the flow to continue without any stoppage. When fluorescent indicator dyes are used to monitor denaturation, another drawback to the stopped flow format is the loss of fluorescent signal due to dye photobleaching while the thermal ramp is being performed.
In a continuous flow format, a melting curve analysis is performed by applying a temperature gradient along the length (direction of flow) of a microchannel in a microfluidic device. If the melting curve analysis requires that the molecules being analyzed be subjected to a range of temperatures extending from a first temperature to a second temperature, the temperature at one end of the microchannel is controlled to the first temperature, and the temperature at the other end of the length is controlled to the second temperature, thus creating a continuous temperature gradient spanning the temperature range between the first and second selected temperatures. A drawback to certain implementations of the continuous flow format is that thermal properties of the molecules in the stream must be measured at multiple points along the temperature gradient to generate the desired melting curve. This makes measurement of thermal properties of the molecules in the stream more complex than in the stopped flow format, where thermal properties of the molecules in the stream can be measured at a single point to generate the desired melting curve.
Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421) and Knight et al. (U.S. Patent Application Publication No. 2007/0231799), each incorporated by reference herein, describe methods, systems, kits and devices for conducting binding assays using molecular melt curves in microfluidic devices. Molecule(s) to be assayed can be flowed through microchannels in the devices where the molecule(s) optionally are exposed to additional molecules constituting, e.g., fluorescence indicator molecules and/or binding partners of the molecule being assayed. The molecules involved are then heated (and/or cooled) and a detectable property of the molecules is measured over a range of temperatures. From the resulting data, a thermal property curve(s) is constructed which allows determination and quantification of the binding affinity of the molecules involved.
Known systems and methods for melt curve analysis suffer from some amount of uncertainty and lack of reproducibility, inter alia, in terms of temperature control and measurement systems. Accordingly, there is a continuing need to improve the usefulness of the melt curve analysis technique by reducing the impact of spatial and temporal temperature fluctuations.